Evaluation of a Rapid, Quantitative Real-Time Pcr Method for Enumeration of Pathogenic Candida Cells in Water

Abstract:

Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaneae) in water (Brinkman et al, 2001). Known numbers of target cells were added to distilled and tap water samples, filtered and the DNA extracted. The assays sensitivities were between one and three cells with a two-fold accuracy around the mean at a 95% confidence interval. In similar tests with surface water samples, the presence of QRT-PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts with resultant reductions in sensitivity but not quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QRT-PCR results and colony forming counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus. With a sample processing time of less than three hours, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking or recreational water and which may be an alternative indicator of fecal pollution._______

Yeasts are a significant component of the micro biota of most natural water systems and can also occur in drinking water distribution systems as a result of their ability to survive treatment practices and become incorporated into biofilms (Woollett and Hedrick, 1970). The majority of these organisms have no known human health effect, however, a small number of species, primarily within the anamorphic genus Candida, are important opportunistic pathogens (Hurley, de Louvi and ).

The importance of pathogenic Candida as agents of nocosomial infections has led to the development of a number of modern molecular diagnostic methods to facilitate their detection and identification in clinical samples (Reiss et al ). Methods based on the polymerase chain reaction (PCR) and DNA hybridization probes have received particular attention (Mannarelli ??; Martin??; Widjojoatmodjo et al 1999 ). The more recent advent of fluorescent probe-based, real-time PCR (RT-PCR) technology (Heid et al 1996) has led to the development of homogeneous methods for detecting these organisms that require relatively short periods of time to perform (Guiver, Levi Oppenhaimer ).

Quantitative Real-Time PCR (QRT-PCR) has been demonstrated to be useful for quantitative analysis of microorganisms (Roe et al 2001), but, to our knowledge, this approach has not been used in the analysis of yeasts in water. Analyses for pathogenic yeasts in drinking or recreational water systems have the potential to expedite the identification of possible health hazards resulting either directly from the presence of these organisms or as indicators of other waterborne pathogens.

The first objective of this study was to develop QRT-PCR technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, for specifically detecting and quantifying six common pathogenic species of Candida including: C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaneae. The second objective was to evaluate a simple and rapid method, using QRT-PCR, for the detection and enumeration of these organisms in different types of water samples. Finally, the technology was tested by comparing this methodology with conventional plating and culturing methods in the analysis of a series of freshwater samples collected from a recreational beach on Lake Michigan.